In article <6gvn32$r6d at net.bio.net>, alcacer at usa.net wrote:
> Hi! I am a stage student in Bourgogne
> University. I have been having problems spheroplasting
> my 48 h. cultures of S.cerevisiae with zymolyase.
> I know spheroplasting younger cultures ( at exponential
> phase )is easier but I would like to achieve spheroplasting
> with 48 h. cells. If anyone could give me a hint of how
> to do that I would be immensely gratefull !! Thank you !!
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Have you tried pre-incubating your cells in spheroplasting buffer
containing high concentrations of beta-mercaptoethanol or DTT before the
enzymic digestion. Reduces the S-S bonds in the cell wall and increases
the "efficiency" of the enzyme. See unit 13:13 in Current Protocols in
Molecular Biology (the "Big Red Book"). This talks about 30 mM DTT and 15
min at room temp. If needed I'm sure you could "incubate" longer. Good
luck.